EFFECTS OF NUCLEOTIDE-INDUCED AND AURODOX-INDUCED CHANGES IN ELONGATION-FACTOR TU CONFORMATION UPON ITS INTERACTIONS WITH AMINOACYL TRANSFER-RNA - A FLUORESCENCE STUDY

The effects of GDP and of aurodox (N-methylkirromycin) on the affinity of elongation factor Tu (EF-Tu) for aminoacyl-tRNA (aa-tRNA) have been quantified spectroscopically by using Phe-tRNAPhe-Fl8, a functionally active analogue of Phe-tRNAPhe with a fluorescein dye covalently attached to the s4U-8 base. The association of EF-Tu•GDP with Phe-tRNAPhe-Fl8 resulted in an average increase of 33% in fluorescein emission intensity. This spectral change was used to monitor the extent of ternary complex formation as a function of EF-Tu•GDP concentration, and hence to obtain a dissociation constant, directly and at equilibrium, for the EF-Tu•GDP-containing ternary complex. The Kd for the Phe-tRNAPhe-Fl8• EF-Tu•GDP complex was found to average 28.5 μM, more than 33000-fold greater than the Kd of the Phe-tRNAPhe-Fl8•EF-Tu•GTP complex under the same conditions. In terms of free energy, the ΔG° for ternary complex formation at 6 °C was −11.5 kcal/mol with GTP and −5.8 kcal/mol with GDP. Thus, the hydrolysis of the ternary complex GTP results in a dramatic decrease in the affinity of EF-Tu for aa-tRNA, thereby facilitating the release of EF-Tu•GDP from the aa-tRNA on the ribosome. Aurodox (200 μM) decreased the Kd of the GDP complex by nearly 20-fold, to 1.46 μM, and increased the Kd of the GTP complex by at least 6-fold. The binding of aurodox to EF-Tu therefore both considerably strengthens EF-Tu•GDP affinity for aa-tRNA and also weakens EF-Tu•GTP affinity for aa-tRNA. As a result, aurodox (and by extension kirromycin) may interfere with EF-Tu-ribosome interactions both/either prior to GTP hydrolysis by interfering with ternary complex recognition and binding by the ribosome and/or following GTP hydrolysis by retarding the release of EF-Tu•GDP from the ribosome. Yet the relatively small magnitude of the aurodox effect upon the affinity of EF-Tu•GDP for aa-tRNA suggests that the strong antibiotic-dependent retardation in EF-Tu•GDP dissociation is mediated primarily by ribosome-EF-Tu interactions. The replacement of GTP by GDP had no detectable effect upon the magnitude of the spectral change that accompanied ternary complex formation, while aurodox caused a small reduction in the size of the spectral change obtained with EF-Tu•GDP. Thus, the EF-Tu-mediated conformational change in the hinge region of the aa-tRNA that is detected by the fluorescent probe appears to be nucleotide-independent and appears to be only slightly altered by the binding of aurodox to EF-Tu•GDP. © 1990, American Chemical Society. All rights reserved.